The development of widespread antibiotic resistance in microbial pathogens has created an urgent medical need for new antimicrobial agents. Instead of relying on derivatives of existing antimicrobial agents, the pharmaceutical industry is looking for novel microbial processes to target in an attempt to create new classes of compounds (Knowles, D. J. C., Trends in Microbiol., 1997,5:379-383).
Genes essential for maintaining an infection in an animal or essential for growth of the pathogen in vitro are good targets for antibiotic development. Traditionally, “essential genes” have been prioritized as good antimicrobial targets. Essential genes are those required for microbial cell growth in vitro and include such genes as those encoding DNA gyrase, ribosomal subunits, and cell wall biosynthetic enzymes. Many of these proteins and cell components have been identified as being encoded by essential genes because there are classic antimicrobial agents shown to inhibit the products of these genes (quinolones, tetracyclines, and “beta”-lactams respectively). Other essential genes have been identified from the characterization of conditional lethal mutants.
With the availability of whole microbial genome sequences, there are now many previously unknown and uncharacterized genes available which may turn out to be essential. The conventional approach for testing if a gene is essential is to attempt making a construct of that organism where the test gene is deleted or inactivated. If the organism can survive with the gene deleted or inactivated, the gene is not considered essential. For example, see Strandén, A. M., Ehlert, K., Labischinski, H., and Berger-Bachi, B., 1997, J. Bacteriol. 179:9-16. However, failure to create a mutant organism with and inactivated or deleted gene does not always mean that the gene is essential. For example, see Okada, K., Minehira, M., Zhu, X., Suzuki, K., Nakagawa, T., Matsuda, H., and Kawamukai, M., 1997, J. Bacteriol. 179:3058-3060. This negative proof for a conclusion may not always be valid. There may be other reasons why the gene deletion or inactivation could not be made.
Recently, virulence factors and genes required for pathogenesis have been suggested as novel targets for antimicrobial agents. Two widely read and referenced techniques, signature tagged mutagenesis (STM; Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E. and Holden, D., 1995, Science 269:400-403) and in vivo expression technology (IVET; Mahan, M. J., Tobias, J. W., Slauch, J. M., Hanna, P. C., Collier, R. J., and Mekalanos, J. J., 1995, PNAS 92:669-673) allow scientists to quickly identify a number of bacterial genes required for pathogenesis or that are induced during host infection. While these genes represent good targets for developing attenuated strains for vaccines, it is not clear if they represent valid targets for inhibition by antimicrobial agents. The critical distinction in this evaluation of potential gene targets is that antimicrobial agents are used to inhibit microbial pathogens after infections are established. If virulence factors or pathogenicity genes are only required to establish the infection, inhibition of these in an established infection would not clear the infection. If, after stopping the synthesis of specific genes, an established infection is cleared, those specific genes are essential for maintaining the infection. Therefore, it would be advantageous to develop a method for turning off an endogenous gene to test if it is essential for growth. Such a method would facilitate the identification of antimicrobial targets which should speed the development of new classes of antimicrobial compounds.
Many of the ideas concerning such systems have been disclosed, see the definitions, theories and descriptions of PCT application PCT/US96/07937, International Publication Number WO 96/40979, published 19 Dec. 1996 (19.12.96). PCT/US96/07937 is hereby incorporated by reference into this document; however, recombinant sequences and the examples disclosed in PCT/US96/07937 are NOT incorporated here.
Also U.S. Pat. No. 5,464,758 disclose many of the mechanisms of the tetracycline-Responsive Promoters. U.S. Pat. No. 5,464,758, published 7 Nov. 1995 is incorporated in part here, the general definitions, theories, principles, concepts, general information about the tetracycline operator (tetO) sequences is incorporated into this document by reference but the sequences disclosed in U.S. Pat. No. 5,464,758 are NOT incorporated into this document. Here Applicant describes a system that works.